This invention describes a novel method for conjugating macromolecules to other molecular entities. Particularly, this invention describes a method for conjugating or derivatizing oligonucleotides and proteins using cycloaddition reactions, such as the Diels-Alder reaction or 1,3-dipolar cycloaddition reactions.
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by Exponential Enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment,xe2x80x9d now abandoned; U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled xe2x80x9cNucleic Acid Ligands,xe2x80x9d now U.S. Pat. No. 5,475,096; U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled xe2x80x9cMethods for Identifying Nucleic Acid Ligands,xe2x80x9d now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which is specifically incorporated by reference herein. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands (also referred to in the art as xe2x80x9captamersxe2x80x9d), each ligand having a unique sequence and property of binding specifically to a desired target compound or molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structure,xe2x80x9d abandoned in favor of U.S. patent application Ser. No. 08/198,670, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligands,xe2x80x9d describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking and/or photoinactiviating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d abandoned in favor of U.S. patent application Ser. No. 08/443,957, now U.S. Pat. No. 5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 5, 1993, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Solution SELEX,xe2x80x9d abandoned in favor of U.S. patent application Ser. No. 08/461,069, now U.S. Pat. No. 5,567,588, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands containing Modified Nucleotides,xe2x80x9d abandoned in favor of U.S. patent application Ser. No. 08/430,709, now U.S. Pat. No. 5,660,985, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. patent application Ser. No. 09/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2-Modified Nucleosides by Intramolecular Nucleophilic Displacement,xe2x80x9d describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,xe2x80x9d now U.S. Pat. No. 5,637,459 and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,xe2x80x9d now U.S. Pat. No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method encompasses complexes of oligonucleotides. U.S. patent application Ser. No. 08/434,465, filed May 4, 1995 entitled xe2x80x9cNucleic Acid Ligand Complexes,xe2x80x9d describes a method for preparing a therapeutic or diagnostic complex comprised of a nucleic acid ligand and a lipophilic compound or a non-immunogenic, high molecular weight compound.
Nucleic acid ligands derived by the SELEX process have been used in diagnostic applications. (See e.g., U.S. patent application Ser. No. 08/487,425, filed Jun. 7, 1995, entitled xe2x80x9cEnzyme Linked Oligonucleotide Assays (ELONAS),xe2x80x9d U.S. patent application Ser. No. 08/479,729, filed Jun. 7, 1995, entitled xe2x80x9cUse of Nucleic Acid Ligands in Flow Cytometry,xe2x80x9d and U.S. patent application Ser. No. 08/628,356, filed Apr. 5, 1996, entitled xe2x80x9cMethod for Detecting a Target Compound in a Substance Using a Nucleic Acid Ligand.xe2x80x9d The full text of the above described patent applications, including but not limited to, all definitions and descriptions of the SELEX process, are specifically incorporated by reference herein in their entirety.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as diagnostic and research reagents and as potential therapeutic agents. There are currently at least three areas of exploration regarding the use of oligonucleotides as pharmaceutical compounds. In the most advanced field, antisense oligonucleotides are used to bind to certain coding regions in an organism to prevent the expression of proteins or to block various cell functions. Additionally, the discovery of RNA species with catalytic functionsxe2x80x94ribozymesxe2x80x94has led to the study of RNA species that serve to perform intracellular reactions that will achieve desired effects. And lastly, the discovery of the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment) (Tuerk and Gold (1990) Science 249:505) has shown that oligonucleotides can be identified that will bind to almost any biologically interesting target.
The use of antisense oligonucleotides as a means for controlling gene expression and the potential for using oligonucleotides as possible pharmaceutical agents has prompted investigations into the introduction of a number of chemical modifications into oligonucleotides to increase their therapeutic activity and stability. Such modifications are designed to increase cell penetration of the oligonucleotides, to stabilize them from nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide analogs in the body, to enhance their binding to targeted RNA, to provide a mode of disruption (terminating event) once sequence-specifically bound to targeted RNA and to improve the pharmacokinetic properties of the oligonucleotides.
Recent research has shown that RNA secondary and tertiary structures can have important biological functions (Tinoco et al. (1987) Cold Spring Harb. Symp. Quant. Biol. 52:135; Larson et al. (1987) Mol. Cell. Biochem. 74:5; Tuerk et al. (1988) Proc. Natl. Acad. Sci. USA 85:1364; Resnekov et al. (1989) J. Biol. Chem. 264:9953). PCT Patent Application Publication WO 91/14436, entitled xe2x80x9cReagents and Methods for Modulating Gene Expression Through RNA Mimicry,xe2x80x9d describes oligonucleotides or oligonucleotide analogs which mimic a portion of RNA able to interact with one or more proteins. The oligonucleotides contain modified internucleoside linkages rendering them nuclease-resistant, have enhanced ability to penetrate cells, and are capable of binding target oligonucleotide sequences.
The use of oligonucleotides as therapeutic and diagnostic agents is growing rapidly with many compounds in human clinical trials. In many of these applications the oligonucleotide is derivatized or conjugated with another molecular entity. These conjugations are typically performed for the purpose of attaching fluorescent dyes or other diagnostic reporter groups or for attaching compounds that modulate the activity or the pharmacokinetic behavior of the oligonucleotide. For example, Smith et al. describe the synthesis of fluorescent dye-conjugated primers for use in fluorescence-based DNA sequence analysis (Smith et al. (1987) Methods Enzymol. 155: 260-301). U.S. Pat. No. 5,650,275 of Pitner et al., describes the use of spectroscopically detectable labeled nucleic acid ligands to determine the presence or absence of a target compound in a sample (see also copending U.S. patent application No. 08/487,425, filed Jun. 7, 1995, entitled xe2x80x9cEnzyme Linked Oligonucleotide Assays (ELONAS),xe2x80x9d U.S. patent application No. 08/479,729, filed Jun. 7, 1995, entitled xe2x80x9cUse of Nucleic Acid Ligands in Flow Cytometry,xe2x80x9d and U.S. patent application No. 08/628,356, filed Apr. 5, 1996, entitled xe2x80x9cMethod for Detecting a Target Compound in a Substance Using a Nucleic Acid Ligandxe2x80x9d). U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled xe2x80x9cNucleic Acid Ligand Complexes,xe2x80x9d describes the use of oligonucleotides conjugated to lipophilic compounds or non-immunogenic, high molecular weight compounds to modulate the activity or pharmokinetic behavior of the oligonucleotides. Conjugation has also been used to make oligonucleotide dimers and to attach oligonucleotides to multimeric platforms. (Jones et al. (1995) J. Med. Chem. 38:2138).
Several chemical methods exist to accomplish such conjugations. (For a review, see Goodchild (1990) Bioconjugate Chemistry 1:165-187). The presence of a chemically reactive functional group, such as an amine or thiol, at the 5xe2x80x2-terminus of an oligonucleotide allows selective attachment of various conjugates, including reporter groups (Smith et al. (1987) Methods Enzymol. 155:260-301; Sproat et al. (1987) Nucleic Acids Res. 15:6181-6196; Mori et al. (1989) Nucleosides and Nucleotides 8:649; Sinha et al. (1988) Nucleic Acids Res. 16:2659) and peptide epitopes (Tung et al. (1991) Bioconjugate Chem. 2:464-465; Bruick et al. (1996) Chem. Biol. 3:49-56). Oligodeoxynucleotides containing a terminal amino functionality have been utilized for the construction of bioconjugates with novel properties. In some of the more common methods of synthesizing these bioconjugates, a primary, aliphatic amino group is incorporated at the 5xe2x80x2-terminus of the oligonucleotide in the final step of the assembly of a synthetic oligonucleotide (Tung et al. (1991) Bioconjugate Chem. 2:464-465; Smith et al. (1987) Methods Enzymol. 155:260-301). A commercial reagent (actually a series of such linkers having various lengths of polymethylene connectors) for linking to the 5xe2x80x2 terminus of an oligonucleotide is 5xe2x80x2-Amino-Modifier C6. These reagents are available from Glen Research Corp (Sterling, Va.). These compounds have been used by Krieg (Krieg et al. (1971) Antisense Res. and Dev.1:161) to link fluorescein to the 5xe2x80x2-terminus of an oligonucleotide. Since many macromolecules of interest are hydrophilic, these reactions generally are done in water, requiring large excesses of reagent to overcome the competing hydrolysis. Usually the amine on the oligonucleotide is added to the terminus of the molecule and must compete with free amine and alcohol on the fully deprotected oligonucleotide if this modification is done post-synthetically.
In another common method of conjugating oligonucleotides to other molecular entities, particularly detector molecules, the molecular entity is converted into a phosphoramidite, which is then added to the free alcohol of the full length oligonucleotide which is attached to a solid support (Theison et al. (1992) Tetrahedron Lett. 33:5033-5036). This method is less than ideal due to the air and water sensitivity of the phosphoramidite, as well as, the fact that the molecule can only be added to the terminus of the oligonucleotide. Furthermore, many detector molecules are not compatible with this method due to the harsh conditions normally needed to fully deprotect and release the oligonucleotide from the support. A third method of conjugating oligonucleotides to other molecules is the coupling of an alkylthio derivatized oligonucleotide with a xcex1-haloacetyl or with a maleimide containing compound. (Jones et al. (1995) J. Med. Chem. 38:2138).
An alternative method for the synthesis of oligodeoxynucleotides terminated by 5xe2x80x2-amino-5xe2x80x2-deoxythymidine has been described (Bruick et al. (1997) Nucleic Acids Res. 25:1309-1310). This method uses a DNA template to direct the ligation of a peptide to an oligonucleotide, in which the peptide is presented by a second oligonucleotide in the form of a reactive thioester-linked intermediate.
The preparation of PEG-oligonucleotide conjugates is described by Goodchild (1990) Bioconjugate Chem. 1:165 and Zalipsky (1995) Bioconjugate Chem. 6:150). The preferred solvent for macromolecule conjugation reactions is an aqueous buffer. Most conjugation chemistry methods must be carried out at high pH and therefore, suffer severely from competing hydrolysis reactions. In addition, most conjugation reactions display poor chemoselectivity.
The preparation of conjugates of macromolecules is not limited to oligonucleotide conjugates. Proteins and peptides play a critical role in virtually all biological processes, functioning as enzymes, hormones, antibodies, growth factors, ion carriers, antibiotics, toxins, and neuropeptides. Proteins and peptides comprise a prominent class of pharmaceuticals. Conjugation of proteins and peptides to detector molecules or other macromolecules such as PEG is also a common practice. Conjugates of oligonucleotides with peptides having specific functions can be useful for various applications. Examples include the use of a nuclear transport signal peptide to direct intracellular trafficking (Eritja et al. (1991) Tetrahedron 47: 4113-4120); a hydrophobic peptide (Juby et al. (1991) Tetrahedron Lett. 32:879-822) or polylysine (Leonetti et al. (1991) Bioconjugate Chem. 1:149-153) to increase cell penetrability, and polylysine to provide multiple attachment sites for nonradioactive reporting probes (Haralambidis et al. (1987) Tetrahedron Lett. 28:5199-5202; Haralambidis et al. (1990) Nucleic Acids Res. 18:493-499).
Cycloaddition reactions can be defined as any reaction between two (or more) moieties (either intra or intermolecular) where the orbitals of the reacting atoms form a cyclic array as the reaction progresses (typically in a concerted fashion although intermediates may be involved) along the reaction coordinate leading to a product. The orbitals involved in this class of reactions are typically xcfx80 systems although certain "sgr" orbitals can also be involved. The number of electrons associated with this type of reaction are of two types; 4n+2 and 4n, were n=1, 2, 3, 4 etc. Typical examples of cycloaddition reactions include Diels-Alder cycloaddition reactions, 1,3-dipolar cycloadditions and [2+2] cycloadditions.
The Diels-Alder reaction, by far the most studied cycloaddition, is a cycloaddition reaction between a conjugated diene and an unsaturated molecule to form a cyclic compound with the xcfx80-electrons being used to form the new "sgr" bonds. The Diels-Alder reaction is an example of a [4+2] cycloaddition reaction, as it involves a system of 4-xcfx80 electrons (the diene) and a system of 2-xcfx80 electrons (the dienophile). The reaction can be made to occur very rapidly, under mild conditions, and for a wide variety of reactants. The Diels-Alder reaction is broad in scope and is well known to those knowledgeable in the art. A review of the Diels-Alder reaction can be found in xe2x80x9cAdvanced Organic Chemistryxe2x80x9d (March. J., ed.) 761-798 (1977) McGraw Hill, N.Y., which is incorporated herein by reference.
It has been discovered that the rate of Diels-Alder cycloaddition reactions is enhanced in aqueous solvents. (Rideout and Breslow (1980) J. Am. Chem. Soc. 102:7816). (A similar effect is also seen with 1,3-dipolar cycloaddition reactions (Engberts (1995) Tetrahedron Lett. 36:5389). This enhancement is presumably due to the hydrophobicity of the diene and dienophile reactants. (Breslow (1991) Acc. Chem. Res. 24:159). This effect extends to intramolecular Diels-Alder reactions. (Blokzijl et al. (1991) J. Am. Chem. Soc. 113:4241). Not only is the reaction rate accelerated in water, but several examples of an increased endo/exo product ratio are also reported. (Breslow and Maitra (1984) Tetrahedron Lett. 25:1239: Lubineau et al. (1990) J. Chem. Soc. Perkin Trans. I, 3011; Grieco et al. (1983) Tetrahedron Lett. 24:1897). Salts which increase the hydrophobic effect in water, such as lithium chloride (Breslow et al. (1983) Tetrahedron Lett. 24:1901) and also monovalent phosphates (Pai and Smith (1995) J. Org. Chem. 60:3731) have been observed to further accelerate the rate of 4+2 cycloadditions.
The synthetic potential of the Diels-Alder reaction in aqueous solvents is gaining increasing attention. It has been demonstrated that simple dienes, such as sodium 3,5-hexadienoate and sodium 4,6-heptadienoate readily undergo Diels-Alder reactions in water with a variety of dienophiles at ambient temperature. (Grieco et al. (1983) J. Org. Chem. 48:3137). The otherwise difficult cycloaddition of dimethyl acetylenedicarboxylate to an electron deficient furan proceeds under very mild conditions in water with very good yields. (Saksena et al. (1993) Heterocycles 35:129). The scope of the reaction has been extended to cycloaddition of iminium salts, generated in situ from an ammonium salt and formaldehyde to dienes. (Grieco and Larsen (1985) J. Am. Chem. Soc. 107:1768). This work inspired the exploration of the corresponding reaction of iminium salts of amino acids with dienes which proceeds with high stereoselectivity. (Grieco et al. (1986) Tetrahedron Lett. 27:1975; Grieco and Bahsas (1987) J. Org. Chem. 52:5745; Waldmann (1989) Liebigs Ann. Chem., 231-238; Waldmann and Braun (1991) Liebigs Ann. Chem., 1045-1048). The scope of this reaction has also been extended to more complex aldehydes by use of lanthanide(III) trifluoromethanesulfonates as catalysts. (Yu et al. (1996) Tetrahedron Lett. 37:2169).
In copending PCT Application Serial No. PCT/US96/16668, filed on Oct. 17, 1996, designating the United States, entitled xe2x80x9cMethod for Solution Phase Synthesis of Oligonucleotidesxe2x80x9d and U.S. application Ser. No. 08/843,820 entitled xe2x80x9cMethod for Solution Phase Synthesis of Oligonucleotides,xe2x80x9d both of which are incorporated herein by reference in their entirety, the Diels-Alder cycloaddition reaction is shown to be an ideal method for anchoring oligonucleotides onto resins. Resins derivatized with a diene or dienophile are reacted with an oligonucleotide derivatized with a dienophile or diene, respectively, to yield the Diels-Alder cycloaddition product. In particular, Diels-Alder reactions between oligonucleotides derivatized with a diene and polymeric resins derivatized with maleimide groups and with phenyl-triazoline-diones (PTAD) are described. The resulting resins can be used as affinity chromatography resins.
The present invention illustrates that cycloaddition reactions, such as the Diels-Alder reaction and 1,3-dipolar cycloaddition reactions, are an ideal replacement for current methods of conjugating macromolecules with other molecular moieties. The Diels-Alder reaction, in particular, is an ideal method for covalently linking large water soluble macromolecules with other compounds as the reaction rate is accelerated in water and can be run at neutral pH. (Rideout and Breslow (1980) J. Am. Chem. Soc. 102:7816). Additionally, the nature of the reaction allows post-synthetic modification of the hydrophilic macromolecule without excess reagent or hydrolysis of the reagent. With respect to conjugation to oligonucleotides, this technology has been aided by the ability to efficiently synthesize 2xe2x80x2-O-diene-nucleosides, which allows the conjugation site to be varied throughout the oligonucleotide or the option of having multiple conjugation sites.
The present invention describes a novel, chemoselective and highly efficient method for derivatizing or conjugating macromolecules with other molecular entities. Specifically, the present invention describes the use of cycloaddition reactions, including but not limited to Diels-Alder reactions, 1,3-dipolar cycloaddition reactions and [2+2] cycloaddition reactions, for the chemoselective and efficient derivatization or conjugation of macromolecules with other molecular entities. Thus, a macromolecule bearing a moiety capable of undergoing a cycloaddition reaction, is reacted with another molecular entity bearing a moiety capable of undergoing a cycloaddition reaction with the moiety attached to the macromolecule to yield via a cycloaddition reaction efficient conjugation of the molecular entity to the macromolecule.
In a preferred embodiment the cycloaddition reaction is a Diels-Alder reaction. Thus, a macromolecule bearing either a diene or dienophile moiety is reacted with another molecular entity bearing either a dienophile or a diene moiety, respectively, to yield via a cycloaddition reaction efficient conjugation of the molecular entity to the macromolecule.
In one embodiment the macromolecule is an oligonucleotide. Thus, an oligonucleotide bearing either a diene modified nucleoside or non-nucleoside phosphate diester group, or a dienophile modified nucleoside or non-nucleoside phosphate diester group is reacted with a molecular entity bearing either a dienophile or a diene moiety. Diels-Alder cycloaddition leads to efficient conjugation of the oligonucleotide with the molecular entity. The molecular entity can be any molecule, including another macromolecule which can be derivatized with a dienophile, diene or other moiety capable of undergoing a cycloaddition reaction. Examples of molecular entities include but are not limited to other macromolecules, polymers or resins, such as polyethylene glycol (PEG) or polystyrene, diagnostic detector molecules, such as fluorescein, coumarin or a metal chelator.
The Diels-Alder cycloaddition between a diene modified oligonucleotide and a dienophile modified oligonucleotide (or any cycloaddition reaction between suitably derivatized oligonucleotides and their reacting partners) results in efficient and specific formation of oligonucleotide homo-dimers and hetero-dimers. In addition, dimers or multimers of oligonucleotides can be prepared efficiently by reaction of two or more diene-modified oligonucleotides with a linker group bearing two or more dienophile moieties. Conventional activated acid linking chemistries do not allow for efficient dimerization or multimerization, since they are limited by competing hydrolysis of the activated acid reagents by water.
This invention includes a reaction scheme for producing a wide variety of conjugated macromolecules using cycloaddition reactions as typified by the Diels-Alder cycloaddition reaction and 1,3-dipolar cycloaddition reactions. The method of this invention can be extended to the conjugation of any macromolecule with another molecular entity, including but not limited to nucleic acids, proteins, peptides carbohydrates, polysaccharides, glycoproteins, lipids, hormones, drugs or prodrugs.
The method of this invention can be extended to all 4n and 4n+2 cycloadditions (where n=1, 2, 3, 4, etc.). This includes, but is not limited to Diels-Alder cycloadditions, 1,3-dipolar cycloadditions, ene cycloaddition reactions, and [2+2] (a 4n type) cycloadditons such as ketene additions and photochemical 2+2 additions.
Also included in this invention are any novel conjugated macromolecules produced by the method of this invention.